Urocortin 2 analogs and uses thereof

ABSTRACT

Disclosed are polypeptides that are analogs of urocortin 2 that have pharmacological activity similar to urocortin 2 but have improved water solubility compared to urocortin 2, and pharmaceutical compositions of the polypeptides of the present invention. Also disclosed are polynucleotides encoding the polypeptides, and methods of treating pathophysiological states employing pharmaceutical compositions of the polypeptides and polynucleotides of the present invention. In addition, disclosed are vectors and host cells that include a nucleic acid encoding a polypeptide of the present invention, and kits that include pharmaceutical compositions of the present invention.

The present application is a continuation of U.S. application Ser. No.14/878,874, filed Oct. 8, 2015, which is a divisional of U.S.application Ser. No. 14/802,812, filed Jul. 17, 2015, now U.S. Pat. No.9,572,866, which is a divisional of U.S. application Ser. No.13/392,828, filed May 4, 2012, abandoned, which is a national phaseapplication under 35 U.S.C. § 371 of International Application No.PCT/US2010/046890, filed Aug. 27, 2010, which claims benefit of U.S.Provisional Application No. 61/237,995, filed Aug. 28, 2009, the entirecontents of each of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates generally to the fields of proteinchemistry, molecular biology, pharmaceutical compositions, andtherapeutics. More particularly, the invention concerns methods,compositions concerning urocortin-2 analogs, and nucleic acids encodingurocortin-2 analogs.

2. Description of Related Art

Corticotropin-releasing factor (CRF) is a 41-amino acid peptide bestknown for its indispensable role in initiating pituitary-adrenalresponses to stress, an effect mediated by type 1 CRF receptors. Inaddition, corticotropin-releasing factor is widely distributed in brain,and participates in the mobilization of complementary autonomic andbehavioral adjustments to a variety of threatening circumstances.Corticotropin releasing factor and its related family of peptides playimportant roles in regulation of the hypothalamic-pituitary-adrenal axis(HPA) under basal and stress conditions. It is also believed thatcorticotropin-releasing factor is also involved in other neuroendocrineand paracrine responses in many tissues. Members of the CRF familyintegrate endocrine, autonomic and behavioral responses to stressors.These peptides may also be implicated in the control of appetite,arousal, and cognitive functions. Severe psychological and physiologicalconsequences can occur as a result of the long term effects of stress,such as anxiety disorders, anorexia nervosa and melancholic depression.

Corticotropin-releasing factor family members mediate their biologicalactions by specifically binding to CRF receptors with high affinities.CRF receptors are G-protein coupled receptors that act through adenylatecyclase and are structurally related to the secretin family. This familyalso includes GRF, VIP, PTH, and the Calcitonin receptor. CRF-R1receptor is distributed throughout the brain and is found in sensory andmotor relay sites. The CRF-R2α is distributed in lateral septum, ventralmedial hypothalamus, nucleus of the solitary tract and the dorsal raphenucleus, which are areas where CRF-R1 is expressed very little or not atall. The CRF-R2β is found mostly in peripheral sites including theheart, blood vessels, gastrointestinal tract, epididymis, lung and skin.The pharmacology of the two types of receptors differs in thatcorticotropin-releasing factor has a low affinity for CRF-R2 but a highaffinity for CRF-R1. Other related peptides such as carp urotensin, frogsauvagine, and urocortin have a high affinity for CRF-R2. CRF-R2knockout mice demonstrate an increased anxiety-like behavior caused byhypersensitivity to stressors.

A mammalian CRF-related neuropeptide, urocortin (Ucn), binds with highaffinity to both known CRF receptor types, whereas CRF is bound in ahighly preferential manner by CRF-R1. Centrally administered urocortinis more potent than CRF in suppressing appetite but less so ingenerating acute anxiety-like effects and generalized behavioralactivation. This has been taken to indicate that urocortin might mediatesome stress-related effects attributed initially to CRF, at least inpart by serving as an endogenous ligand for CRF-R2. Urocortin has beenproposed as a therapeutic agent in heart disease (see, e.g., Raddino etal., 2007; Davidson et al., 2009).

Urocortin 2 (Ucn 2), one of the CRF peptide family members, is thoughtto be an endogenous ligand for CRF type 2 receptor (CRF-R2). The rolesof urocortin 2 in the body are diverse, and include involvement inaffective disorders, stimulation of the immune system, andcardioprotection (Lawrence and Latchman, 2006). Therapeutic applicationof urocortin 2 is limited because of the limited water solubility of theurocortin 2 peptide. Therefore, there is the need for active urocortin 2analogs that have improved water solubility.

SUMMARY OF THE INVENTION

The present invention is in part based on the identification of analogsof urocortin 2 that have the surprising and unexpected ability tostimulate intracellular cAMP production in cells in a manner similar tourocortin 2, yet have improved water solubility compared to urocortin 2.Improved water solubility will lead to greater therapeutic applicationof urocortin 2 analogs in the treatment and prevention of diseases andhealth-related conditions involving the hypothalamic-pituitary-adrenalaxis.

Some embodiments of the present invention include urocortin analogs thatare isolated polypeptides of formula (I):

X₁-X₂-X₃-X₄-X₅-X₆  (I)

wherein X₁ is either absent or a polypeptide having 1-200 residues; X₂is absent or 1-20 amino acid residues that are each individuallyselected from either arginine (R) or lysine (K); X₃ is SEQ ID NO:1, SEQID NO:19, SEQ ID NO:21, or SEQ ID NO:22; X₄ is either absent, G, GH, orGHC; X₅ is absent or 1-20 amino acid residues that are each individuallyselected from either arginine (R) or lysine (K); X₆ is either absent ora polypeptide having 1-200 residues.

Some embodiments of the present invention include the proviso that if X₃is SEQ ID NO:1, X₄, X₅, X₆ are all absent, then X₂ is not absent,further provided that if X₃ is SEQ ID NO:19, X₅ is absent and X₆ isabsent, then X₄ is G, GH, or GHC. In particular embodiments, X₃ is SEQID NO: 1. In further particular embodiments, X₃ is SEQ ID NO:19. Infurther particular embodiments, X₂ is arginine (R).

In particular embodiments, X₁ and X₂ are absent, X₃ is SEQ ID NO:1, X₄is glycine, and X₆ is absent.

In other embodiments, X₁ is absent, X₃ is SEQ ID NO:1, X₄ is absent, X₅is absent, X₆ is absent, and the polypeptide is further defined as beingamidated at the C-terminus.

In further embodiments, X₁ and X₂ are absent, X₃ is SEQ ID NO:19, X₄ isglycine, and X₆ is absent.

In other embodiments, the polypeptide of formula (I) is further definedas being amidated at the C-terminus. Amidation can be by any methodknown to those of ordinary skill in the art.

X₂ and X₅ may be a consecutive series of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids that are either R and K.In particular embodiments, X₂ and/or X₅ include 2 amino acid residues.In specific embodiments, the amino acid residue is arginine (R).

Other embodiments of the present invention include any of the foregoingpeptides that are amidated at the C-terminus. Some embodiments include aC residue at the N-terminus and a C residue at the C-terminus.

Other embodiments concern urocortin 2 or a peptide derived fromurocortin 2 that is amidated at the C-terminus. Other analogs ofurocortin 2 include any peptide derived from a prohormone of urocortin 2that is amidated at the C-terminus. In some embodiments the urocortin 2analog is a full-length prohormone of urocortin 2 that is amidated atthe C-terminus. The prohormone of urocortin 2 or urocortin may be fromany species. In particular embodiments, the urocortin 2 or prohormone ofurocortin 2 is from human or mouse. In specific embodiments, theurocortin analog is SEQ ID NO:9 or SEQ ID NO:12.

Other embodiments of the present invention include analogs of urocortin2 that are isolated polypeptides of formula I: Z₁-Z₂, where Z₁ is (i)one amino acid selected from the group of amino acids consisting of Rand K or (ii) a consecutive series of amino acids selected from thegroup consisting of R and K; and Z₂ comprises a urocortin 2 orprohormone of urocortin 2 amino acid sequence. For example, in someembodiments Z₂ is IVLSLDVPIGLLQILLEQARARAAREQATTNARILARV (SEQ ID NO:1),SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:22, provided that the carboxyterminus of Z₁ is covalently attached to the N-terminus of SEQ ID NO:1.The analog may optionally be amidated at the C-terminus.

Other embodiments of the present invention include analogs thatcomprises a urocortin 2 or prohormone of urocortin 2 amino acid sequencethat includes G, GH, or GHC attached to the C-terminus of the urocortin2 or prohormone of urocortin 2 amino acid sequence.

A table of amino acid abbreviations is provided in the specificationbelow; the single letter abbreviations for amino acids will be usedthroughout this disclosure. The isolated polypeptides of the presentinvention a consecutive series of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more aminoacids or any range of amino acids derivable therein. For example, Z₁ maybe 2-30 consecutive amino acids in length, 2-20 consecutive amino acidsin length, 2-10 consecutive amino acids in length, or 2-5 consecutiveamino acids in length. In particular embodiments, Z₁ consists of oneamino acid selected from the group consisting of R and K. In a specificembodiment, Z₁ is R (arginine). Z₂ may include one or a series ofconsecutive amino acids attached to the C-terminus. For example, theC-terminus may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, 1000 or any range of amino acids derivable therein. Forexample, there may be 2-500, 2-400, 2-300, 2-200, 2-100, 2-50, 2-20, or2-10 consecutive amino acids attached to the C-terminus of SEQ ID NO:1.In specific embodiments, Z₁ is SEQ ID NO:1 (i.e., with no amino acidsattached to the C-terminus of SEQ ID NO:1). In particular embodiments,the isolated polypeptide is SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

In some embodiments, the C-terminus or the N-terminus of the polypeptideis protected.

In particular embodiments, the C-terminus of any of the foregoingpolypeptides is amidated. Binding of any moiety known to form an amidewhen reacted with the carboxy terminus of a polypeptide is contemplatedby the present invention.

In some embodiments, the isolated polypeptide of the present inventionmay have a protected C-terminus or a protected N-terminus. Thus, forexample, a protecting group may be attached to the N-terminus orC-terminus of the polypeptide of the present invention. In someembodiments, the C-terminus is amidated. In some embodiments, an aminoprotecting agent is used to protect the N-terminus.

Some embodiments of the instant invention include analogs of urocortinas set forth herein that have an acylated N-terminus. This proteinacylation may be used to link a molecule such as fatty acid at theN-terminus of the protein to protect the polypeptide from enzymaticdegradation or to change various properties of the protein such as itshydrophilicity/hydrophobicity. These modifications may be used to alterthe duration or bioavailability of the protein in vivo.

In some embodiments, the isolated polypeptides of the present inventioncan be conjugated to complexing agents for radionuclides. As mentioned,conjugation between the complexing agent and the polypeptides set forthherein may take place via any method and chemical linkage known to thoseof skill in the art. A protecting group may be removed prior toconjugation to a complexing agent. The complexing agent may be achelator. The radionuclide may be any radionuclide known to those ofordinary skill in the art. In non-limiting examples, the metal ion maybe selected from the group consisting of a technetium ion, a copper ion,an indium ion, a thallium ion, a gallium ion, an arsenic ion, a rheniumion, a holmium ion, a yttrium ion, a samarium ion, a selenium ion, astrontium ion, a gadolinium ion, a bismuth ion, an iron ion, a manganeseion, a lutecium ion, a cobalt ion, a platinum ion, a calcium ion and arhodium ion. The radionuclide-labeled analogs can be used for imagingsuch as scintigraphy.

The isolated polypeptides of the present invention may be modified tocontain a label, such as radioactive elements, enzymes, chemicals thatfluoresce when exposed to ultraviolet light, and others. A number offluorescent materials are known and can be utilized as labels. Theseinclude, for example, fluorescein, rhodamine, auramine, Texas Red, AMCAblue and Lucifer Yellow. The resulting labeled urocortin 2 analogs canbe used to identify cells expressing CRF receptors for biologicalassays. Alternatively, the polypeptides set forth herein may be linkedto a toxin molecule. The resulting toxic conjugate can be used for thetargeted destruction of CRF receptor-bearing cells.

In specific embodiments, the polypeptide includes SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQID NO:17, or SEQ ID NO:18.

The present invention also contemplates chimeric polypeptide thatinclude a first amino acid sequence that is a recombinant urocortin 2analog and a second amino acid sequence, wherein the second amino acidsequence is a therapeutic amino acid sequence. In some embodiments, thesecond amino acid sequence enhances the bioavailability of the firstamino acid sequence. For example the second amino acid sequence mayenhance bioavailability across the blood-brain barrier. For example, thesecond amino acid sequence may be selected from the group consisting ofa low density lipoprotein receptor binding domain of apolipoprotein B,an Fc amino acid sequence, or a toxin. Non-limiting examples of toxinsinclude gelonin, dodecandrin, tricosanthin, tricokirin, bryodin,mirabilis antiviral protein, barley ribosome-inactivating protein(BRIP), pokeweed antiviral protein (PAPs), saporin, luffin, momordin,ricin, abrin, diphtheria toxin A, pertussis toxin A subunit, E. colienterotoxin toxin A subunit, cholera toxin (CTX) and Pseudomonas toxinc-terminal. In some embodiments, the chimeric polypeptide is a fusionprotein encoded by a single polynucleotide. Polynucleotides encoding thechimeric polypeptides set forth herein are also contemplated. Methods oftreating disease, compositions, and kits employing the chimericpolypeptides and polynucleotides of the present invention are alsocontemplated.

Further embodiments of the present invention include a recombinantnucleic acid that includes a nucleic acid segment encoding any of thepolypeptides of the present invention. In specific embodiments, thepolypeptide includes SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18. Inparticular embodiments, X₆ is —OH.

Other aspects of the invention concern recombinant nucleic acidsencoding any of the aforementioned polypeptides. The nucleic acids mayoptionally encode one or more additional amino acid residues. Thenucleic acid may optionally be comprised in a vector. Non-limitingexamples of vectors include nanoparticles comprising a lipid, viralvectors, and cells. The vector may further include regulatory elementsnecessary for expression of said nucleic acid sequence in a cell. Inparticular embodiments, the vector may be a viral vector. For example,the viral vector may be an adenoviral vector, a lentiviral vector, or anadeno-associated viral vector. The vector also includes host cellstransfected with vectors as set forth herein. Non-limiting examples ofhost cells include a bacterial cell, a mammalian cell, a plant cell, oran insect cell. As used herein, the term “host” is meant to include notonly prokaryotes but also eukaryotes such as yeast, plant and animalcells. A recombinant DNA molecule or gene that encodes a polypeptide ofthe present invention can be used to transform a host using any of thetechniques commonly known to those of ordinary skill in the art.Prokaryotic hosts may include E. coli, S. tymphimurium, S. marcescensand B. subtilis. Non-limiting examples of eukaryotic hosts includeyeasts such as P. pastoris, mammalian cells and insect cells.

Other aspects of the invention concern pharmaceutical compositions thatinclude any of the aforementioned polypeptides or recombinant nucleicacids, and a pharmaceutically acceptable carrier. Non-limiting examplesof pharmaceutically acceptable carriers include water or normal saline.

The present invention also concerns methods of treating apathophysiological state (disease or health-related condition) in asubject, involving administering to the subject a pharmaceuticalcomposition that includes any of the polypeptides, chimericpolypeptides, or polynucleotides as set forth herein and apharmaceutically acceptable carrier. The subject may be any subject. Inparticular embodiments, the subject is a mammal. Non-limiting examplesof mammals include a human, a primate, a horse, a cow, a sheep, a pig, adog, a cat, a rat, or a mouse. In specific embodiments, the mammal is ahuman. The human may be a patient with a pathophysiological state or atrisk of developing a pathophysiological state. The pathophysiologicalstate is any pathophysiological state known to those of ordinary skillin the art. In some embodiments, the pathophysiological state is adisease or health-related condition that involves the HPA. Non-limitingexamples of pathophysiological states include affective disorders,disorders of elevated body temperature, appetite dysfunction, obesity,abnormalities of glucose metabolism, heart disease, stress, anxiety, andundesirably low levels of ACTH secretion. Non-limiting examples ofaffective disorders include depression, manic-depressive disease, andschizophrenia. Non-limiting examples of disorders of elevated bodytemperature include infections and malignancies. Non-limiting examplesof heart disease include congestive heart failure, myocardialinfarction, angina, and arrhythmias. Non-limiting examples ofabnormalities of glucose metabolism include impaired glucose tolerance,hyperglycemia, and diabetes.

Treatment of any pathophysiological state wherein urocortin analoguesare known or suspected to be of value is contemplated by the presentinvention. Non-limiting examples of pathophysiological states includehigh body temperature, appetite dysfunction, congestive heart failure,stress, anxiety, and undesirably low levels of ACTH secretion.

Host cells transfected with a vector of the present invention are alsocontemplated by the present invention. For example, the host cell may bea bacterial cell, a mammalian cell, a plant cell, or an insect cell.

Kits that include a sealed container that includes a pharmaceuticallyacceptable composition of any of the polypeptides or nucleic acids ofthe present invention are also contemplated. The kit may include anynumber of additional components as discussed in the specification below.In particular embodiments, the kit further includes a secondarypharmaceutical agent that can be applied in the treatment of apathophysiological state.

It is specifically contemplated that any limitation discussed withrespect to one embodiment of the invention may apply to any otherembodiment of the invention. Furthermore, any composition of theinvention may be used in any method of the invention, and any method ofthe invention may be used to produce or to utilize any composition ofthe invention.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativeare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device and/ormethod being employed to determine the value.

As used herein the specification, “a” or “an” may mean one or more,unless clearly indicated otherwise. As used herein in the claim(s), whenused in conjunction with the word “comprising,” the words “a” or “an”may mean one or more than one. As used herein “another” may mean atleast a second or more.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention is partly based on the identification of analogsof urocortin 2 that have a similar ability to stimulate cAMP productionin cells as compared to urocortin 2, but which are surprisingly andunexpectedly more water soluble than urocortin 2. This increased watersolubility allows for increased pharmaceutical application of theseagents, and the improved treatment of a variety of pathophysiologicalstates that implicate the HPA axis.

A. Urocortin 2 Analogs

The present invention includes polypeptides that are analogs ofurocortin 2 as discussed above. All amino-acid residue sequences arerepresented herein by formulae whose left and right orientation is inthe conventional direction of amino-terminus to carboxy-terminus and bysingle letter abbreviations. Table 1 sets forth a table of the commonamino acids and abbreviations known to those of ordinary skill in theart.

TABLE 1 Amino Acids 3-Letter Single Letter Amino Acids AbbreviationAbbreviation Alanine Ala A Cysteine Cys C Aspartic acid Asp D Glutamicacid Glu E Phenylalanine Phe F Glycine Gly G Histidine His H IsoleucineIle I Lysine Lys K Leucine Leu L Methionine Met M Asparagine Asn NProline Pro P Glutamine Gln Q Arginine Arg R Serine Ser S Threonine ThrT Valine Val V Tryptophan Trp W Tyrosine Tyr Y

It should be noted that a dash at the beginning or end of an amino acidresidue sequence indicates a peptide bond to a further sequence of oneor more amino-acid residues.

The amino acids described herein are preferred to be in the “L” isomericform. However, residues in the “D” isomeric form can be substituted forany L-amino acid residue, as long as the desired functional property ofimmunoglobulin binding is retained by the polypeptide. NH.sub.2 at theamino-terminus refers to the free amino group present at the aminoterminus of a polypeptide. —OH at the carboxy terminus refers to thefree carboxy group present at the carboxy terminus of a polypeptide.NH.sub.2 at the carboxy terminus refers to a C-terminal amide present atthe carboxy terminus of a polypeptide.

Nonstandard amino acids may be incorporated into proteins by chemicalmodification of existing amino acids or by artificial synthesis of aprotein. A nonstandard amino acid refers to an amino acid that differsin chemical structure from the twenty standard amino acids encoded bythe genetic code. Post-translational modification in vivo can also leadto the presence of a nonstandard or amino acid derivative in a protein.The N-terminal NH.sub.2 and C-terminal COOH groups of a protein can alsobe modified by natural or artificial post-translational modification ofa protein.

Various embodiments of the present invention pertain to methods fortreating or preventing a pathophysiological state in a subjectcomprising a pharmaceutical composition comprising a polypeptide ornucleic acid encoding a polypeptide of the present invention as setforth herein.

As used herein, the term “polypeptide” is a consecutive amino acidsegment of greater than two amino acids in length. As set forth herein,the polypeptides of the present invention comprise SEQ ID NO:1. Thepolypeptides set forth herein can include one or more amino acidsattached to the N-terminus of SEQ ID NO:1, wherein the amino acids arehydrophilic amino acids as discussed above. Further, the polypeptidesset forth herein can include one or more consecutive amino acidsattached to the C-terminus of SEQ ID NO:1. For example, the polypeptidecan be a polypeptide that includes 4, 5, 10, 15, 20, 25, 30, 50, 100,200, 300, 400, 500, 1000 or any number of consecutive amino acidsattached to the C-terminus of SEQ ID NO:1. The polypeptide may include0, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 100, or moreconsecutive hydrophilic amino acids attached to the N-terminus of SEQ IDNO:1. One of ordinary skill in the art would understand how to generatea polypeptides of the present invention in view of the disclosure usingany of a number of experimental methods well-known to those of skill inthe art.

Also encompassed in the present invention are polypeptide variants ofthe polypeptides as set forth herein. For example, the polypeptidevariants may include a certain amount of sequence identity compared tothe polypeptides of the present invention. Thus, for example,polypeptide variants may be variants that have an amino acid identity of80%, 85%, 90%, 95%, 98%, or 99% or more, or any range of amino acididentify derivable therein, with (1) a polypeptide that consists of SEQID NO:1 with a R (arginine) moiety attached to the N-terminus of SEQ IDNO:1, (2) SEQ ID NO:2, (3) SEQ ID NO:3, or (4) SEQ ID NO:4.

The present invention may utilize polypeptides purified from a naturalsource or obtained from recombinantly-produced material. Those ofordinary skill in the art would know how to produce these polypeptidesfrom recombinantly-produced material. This material may use the 20common amino acids in naturally synthesized proteins, or one or moremodified or unusual amino acids. Generally, “purified” will refer to acomposition that has been subjected to fractionation to remove variousother proteins, polypeptides, or peptides. Purification may besubstantial, in which the polypeptide or equivalent is the predominantspecies, or to homogeneity, which purification level would permitaccurate degradative sequencing.

Amino acid sequence mutants also are encompassed by the presentinvention, and are included within the definition of “polypeptidevariants.” Amino acid sequence variants of the polypeptide can besubstitutional mutants or insertional mutants. Insertional mutantstypically involve the addition of material at a non-terminal point inthe peptide. This may include the insertion of a few residues, or simplya single residue. The added material may be modified, such as bymethylation, acetylation, and the like. Alternatively, additionalresidues may be added to the N-terminal or C-terminal ends of thepeptide.

Amino acid substitutions are generally based on the relative similarityof the amino acid side-chain substituents, or for example, theirhydrophobicity, hydrophilicity, charge, size, and the like. An analysisof the size, shape and type of the amino acid side-chain substituentsreveals that arginine, lysine and histidine are all positively chargedresidues; that alanine, glycine and serine are all a similar size; andthat phenylalanine, tryptophan and tyrosine all have a generally similarshape. Therefore, based upon these considerations, arginine, lysine andhistidine; alanine, glycine and serine; and phenylalanine, tryptophanand tyrosine; are defined herein as biologically functional equivalents.

B. Chimeric Proteins

The present invention also concerns chimeric proteins that include afirst amino acid sequence that is a urocortin 2 analog and a secondamino acid sequence attached to the N or C-terminus of the urocortin 2analog. The second amino acid sequence may be any amino acid sequencethat includes 2 or more amino acid residues. In some embodiments, thesecond amino acid sequence is a therapeutic peptide or polypeptide.Examples include amino acid sequences that enhance biological activityof the urocortin 2 analog. Examples include amino acid sequences thatfacilitate penetration of the urocortin 2 analog across the blood-brainbarrier. One example is the low-density lipoprotein receptor bindingdomain of apolipoprotein B (Spencer and Verma, 2007). In specificembodiments, a lentivirus vector system is used to delivery a fusionprotein that includes a urocortin 2 analog fused to a low-densitylipoprotein receptor binding domain of apolipoprotein A. The amino acidsequence of the low-density lipoprotein receptor binding domain ofapolipoprotein A includes amino acids 3371-3409 of human ApoB (GenBankAccession Number AAH51278), herein after SEQ ID NO:20. Other specificexamples of therapeutic amino acid sequences that facilitatetranslocation of amino acid sequences across cell membranes include theHIV TAT sequence (Nagahara et al., 1998), the third helix of theAntennapedia homeodomain (Antp) (Derossi et al., 1994), and the HSV-1structural protein VP22 (Elliott and O'Hare, 1997). In otherembodiments, the second amino acid sequence is an agent that increasesduration of action of the urocortin-2 polypeptide. One non-limitingexample is an amino acid sequence encoding Fc or a fragment thereof.Other class of possible second amino acid sequences include agents thatcan kill cells. For example, the second amino acid sequence may be atoxin, such as gelonin, dodecandrin, tricosanthin, tricokirin, bryodin,mirabilis antiviral protein, barley ribosome-inactivating protein(BRIP), pokeweed antiviral proteins (PAPs), saporins, luffins,momordins, ricin, abrin, diphtheria toxin A, pertussis toxin A subunit,E. coli enterotoxin toxin A subunit, cholera toxin (CTX) and Pseudomonastoxin c-terminal.

The two moieties of the chimeric protein produced by synthetic orrecombinant methods may be conjugated by linkers according to methodswell known in the art (Brinkmann and Pastan, 1994). As used herein, a“linker” is a chemical or peptide or polypeptide that links a firstamino acid sequence with a second amino acid sequence. Non-limitingexamples of linkers include flexible polylinkers, such as one composedof a pentamer of four consecutive glycine resides with a serine residueat the C-terminus. Such a linker may be repeated 1 or more times. Anyother linker known to those of ordinary skill in the art is contemplatedby the present invention.

It is contemplated that cross-linkers may be implemented to fuse thefirst amino acid sequence and the second amino acid sequence.Cross-linking reagents are used to form molecular bridges that tietogether functional groups of two different molecules. To link twodifferent polypeptides in a step-wise manner, hetero-bifunctionalcross-linkers can be used that eliminate unwanted homopolymer formation.Bifunctional cross-linking reagents have been extensively used for avariety of purposes including preparation of affinity matrices,modification and stabilization of diverse structures, identification ofbinding sites, and structural studies.

In some embodiments, the chimeric protein is further defined as a fusionprotein. A “fusion protein” as used herein refers to a polypeptideencoded by a single recombinant polynucleotide encoding the chimericprotein. The chimeric polypeptides set forth herein may comprises asequence of any number of additional amino acid residues at either theN-terminus or C-terminus of the chimeric polypeptide.

“Chemical derivative” refers to a subject polypeptide having one or moreresidues chemically derivatized by reaction of a functional side group.Such derivatized polypeptides include, for example, those in which freeamino groups have been derivatized to form amine, p-toluene sulfonylgroups, carbobenzoxy groups, t-butyloxycarbonyl groups, chioroacetylgroups, or formyl groups. Free carboxyl groups may be derivatized toform salts, methyl and ethyl esters or other types of esters orhydrazides. Chemical derivatives may include those peptides whichcontain one or more naturally occurring amino acids derivatives of thetwenty standard amino acids. For example, 4-hydroxyproline may besubstituted for serine; and ornithine may be substituted for lysine.Peptides embraced by the present invention also include peptides havingone or more residue additions and/or deletions relative to the specificpeptide whose sequence is shown herein, so long as the modified peptidemaintains the requisite biological activity.

Additional information regarding urocortin 2 and urocortins can be foundin U.S. Patent Application Publication Nos. 20080161235, 20070191592,20070042954, 20050191650, and 20030032587, and U.S. Pat. Nos. 7,507,794,7,488,865, 7,459,427, 7,223,846, 7,141,546, 6,838,274, 6,353,152, and6,214,797, each of the foregoing of which is herein specificallyincorporated by reference in its entirety.

C. Polynucleotides Encoding Urocortin 2 Analogs

In certain embodiments the present invention concerns polynucleotidesencoding urocortin 2 or analogs of urocortin 2, and uses of suchpolynucleotides in methods as set forth herein.

The polynucleotide may include additional nucleic acid sequences that donot encode a urocortin 2 analog. The polynucleotides may be derived fromgenomic DNA, i.e., cloned directly from the genome of a particularorganism. The polynucleotide may be a DNA or a RNA.

In some embodiments, the polynucleotides may be complementary DNA(cDNA). cDNA is DNA prepared using messenger RNA (mRNA) as a template.Thus, a cDNA does not contain any interrupted coding sequences andusually contains almost exclusively the coding region(s) for thecorresponding protein. In other embodiments, the polynucleotide may beproduced synthetically.

It may be advantageous to combine portions of the genomic DNA with cDNAor synthetic sequences to generate specific constructs. For example,where an intron is desired in the ultimate construct, a genomic clonewill need to be used. Introns may be derived from other genes inaddition to urocortin 2. The cDNA or a synthesized polynucleotide mayprovide more convenient restriction sites for the remaining portion ofthe construct and, therefore, would be used for the rest of thesequence.

The polynucleotides encoding urocortin 2 analogs may benaturally-occurring homologous polynucleotide sequences from otherorganisms. A person of ordinary skill in the art would understand thatcommonly available experimental techniques can be used to identify orsynthesize polynucleotides encoding urocortin analogs. The presentinvention also encompasses chemically synthesized mutants of thesesequences.

Another kind of sequence variant results from codon variation. Becausethere are several codons for most of the 20 normal amino acids, manydifferent DNAs can encode a urocortin 2 analog. One of ordinary skill inthe art would understand these variants.

Allowing for the degeneracy of the genetic code, sequences that havebetween about 50% and about 75%, or between about 76% and about 99% ofnucleotides that are identical to urocortin 2 may be considered asurocortin 2 analogs of the present invention. Sequences that are withinthe scope the polynucleotides used in the methods set forth herein arethose that are capable of base-pairing with a polynucleotide segment setforth above under intracellular conditions.

As stated above, the polynucleotides employed in the methods set forthherein may be full length genomic or cDNA copies, or large fragmentsthereof. The present invention also may employ shorter oligonucleotides.Sequences of 12 bases long should occur only once in the human genomeand, therefore, suffice to specify a unique target sequence.

In certain embodiments, one may wish to employ constructs which includeother elements, for example, those which include C-5 propynepyrimidines. Oligonucleotides which contain C-5 propyne analogues ofuridine and cytidine have been shown to bind RNA with high affinity(Wagner et al., 1993).

For those urocortin 2 analogs with a C-terminal amide, the correspondingnucleic acid sequence would be a nucleic acid sequence encoding Gly-X orGly-X-X, where X is either Arg or Lys.

In some embodiments, the polynucleotides set forth herein encode achimeric protein that includes a first amino acid sequence that is aurocortin 2 analog and a second amino acid sequence, as discussed above.

D. Pharmaceuticals and Methods for the Treatment of Disease

In additional embodiments, the present invention concerns formulation ofone or more of the polynucleotides and/or polypeptides disclosed hereinin pharmaceutically-acceptable carriers for administration to a cell,tissue, animal, patient, or subject either alone, or in combination withone or more other modalities of therapy.

Aqueous pharmaceutical compositions of the present invention will havean effective amount of a polypeptide or polynucleotide of the presentinvention. Such compositions generally will be dissolved or dispersed ina pharmaceutically acceptable carrier or aqueous medium. An “effectiveamount,” for the purposes of therapy, is defined as that amount thatcauses a clinically measurable difference in the condition of thesubject. This amount will vary depending on the substance, the conditionof the patient, the type of treatment, etc.

The phrases “pharmaceutically or pharmacologically acceptable” refer tomolecular entities and compositions that do not produce a significantadverse, allergic or other untoward reaction when administered to ananimal, or human. As used herein, “pharmaceutically acceptable carrier”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredients, its use inthe therapeutic compositions is contemplated.

In addition to the compounds formulated for parenteral administration,such as those for intravenous or intramuscular injection, otherpharmaceutically acceptable forms include, e.g., tablets or other solidsfor oral administration; time release capsules; and any other formcurrently used, including creams, lotions, inhalants and the like.

The active compounds of the present invention will often be formulatedfor parenteral administration, e.g., formulated for injection via theintravenous, intramuscular, subcutaneous, or even intraperitonealroutes. The preparation of an aqueous composition that contains apolypeptide or polynucleotide of the present invention alone or incombination with a conventional therapeutic agent as active ingredientswill be known to those of skill in the art in light of the presentdisclosure. Typically, such compositions can be prepared as injectables,either as liquid solutions or suspensions; solid forms suitable forusing to prepare solutions or suspensions upon the addition of a liquidprior to injection can also be prepared; and the preparations can alsobe emulsified.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In many cases, the form must be sterile and must be fluidto the extent that easy syringability exists. It must be stable underthe conditions for manufacture and storage and must be preserved againstthe contaminating action of microorganisms, such as bacteria and fungi.

The carrier also can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousother ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle whichcontains the basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum-drying and freeze-drying techniques which yield apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above,with even drug release capsules and the like being employable.

The composition can be administered to the subject using any methodknown to those of ordinary skill in the art. For example, apharmaceutically effective amount of the composition may be administeredintravenously, intracerebrally, intracranially, intrathecally, into thesubstantia nigra or the region of the substantia nigra, intradermally,intraarterially, intraperitoneally, intralesionally, intratracheally,intranasally, topically, intramuscularly, intraperitoneally,subcutaneously, orally, locally, inhalation (e.g., aerosol inhalation),injection, infusion, continuous infusion, localized perfusion bathingtarget cells directly, via a catheter, via a lavage, in cremes, in lipidcompositions (e.g., liposomes), or by other method or any combination ofthe forgoing as would be known to one of ordinary skill in the art(Remington's, 1990).

In particular embodiments, the composition is administered to a subjectusing a drug delivery device. Any drug delivery device is contemplatedfor use in delivering a pharmaceutically effective amount of apolypeptide or polynucleotide of the present invention.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline, mannitol or glucose.These particular aqueous solutions are especially suitable forintravenous, intramuscular, subcutaneous and intraperitonealadministration. In this connection, sterile aqueous media which can beemployed will be known to those of skill in the art in light of thepresent disclosure. For example, one dosage could be dissolved in 1 mLof isotonic NaCl solution and either added to 1000 mL of hypodermoclysisfluid or injected at the proposed site of infusion, (see for example,“Remington's Pharmaceutical Sciences” (1980)). Some variation in dosagewill necessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject.

In certain aspects of the methods of the invention, the route thetherapeutic composition is administered may be by parenteraladministration. The parenteral administration may be intravenousinjection, subcutaneous injection, intramuscular injection,intramedullary injection, ingestion or a combination thereof. In certainaspects, the composition comprising a polypeptide or polynucleotide ofthe present invention is administered from about 0.1 to about 10microgram/kg/body weight per dose. In certain aspects, the compositionis administered from about 1 to about 5 microgram/kg/body weight perdose. In certain aspects, the composition is administered from about 1.2to about 3.6 microgram/kg/body weight per dose. In certain aspects, thecomposition is administered from about 1.2 to about 2.4microgram/kg/body weight per dose. In preferred aspects, the amount ofpolypeptide or polynucleotide of the present invention administered perdose may be about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2,about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5,about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about3.2, about 3.3, about 3.4, about 3 5, about 3.6, about 3.7, about 3.8,about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1,about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4,about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7,about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0,about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about9.7, about 9.8, about 9.9, about 10.0, or more micrograms/kg/body.

Formulation of pharmaceutically-acceptable excipients and carriersolutions is well-known to those of skill in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., oral, parenteral, intravenous, intranasal, andintramuscular administration and formulation.

The term “alimentary delivery” refers to the administration, directly orotherwise, to a portion of the alimentary canal of an animal. The term“alimentary canal” refers to the tubular passage in an animal thatfunctions in the digestion and absorption of food and the elimination offood residue, which runs from the mouth to the anus, and any and all ofits portions or segments, e.g., the oral cavity, the esophagus, thestomach, the small and large intestines and the colon, as well ascompound portions thereof such as, e.g., the gastro-intestinal tract.Thus, the term “alimentary delivery” encompasses several routes ofadministration including, but not limited to, oral, rectal, endoscopicand sublingual/buccal administration. A common requirement for thesemodes of administration is absorption over some portion or all of thealimentary tract and a need for efficient mucosal penetration of theagent so administered.

In certain applications, the pharmaceutical compositions disclosedherein may be delivered via oral administration to an animal, patient,or subject. As such, these compositions may be formulated with an inertdiluent or with an assimilable edible carrier, or they may be enclosedin hard- or soft-shell gelatin capsule, or they may be compressed intotablets, or they may be incorporated directly with the food of the diet.

The active components may even be incorporated with excipients and usedin the form of ingestible tablets, buccal tables, troches, capsules,elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et al.,1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515; 5,580,579 and5,792,451, each specifically incorporated herein by reference in itsentirety). The tablets, troches, pills, capsules and the like may alsocontain the following: a binder, as gum tragacanth, acacia, cornstarch,or gelatin; excipients, such as dicalcium phosphate; a disintegratingagent, such as corn starch, potato starch, alginic acid and the like; alubricant, such as magnesium stearate; and a sweetening agent, such assucrose, lactose or saccharin may be added or a flavoring agent, such aspeppermint, oil of wintergreen, or cherry flavoring. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier. Various other materials may be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills, or capsules may be coated with shellac,sugar, or both. A syrup of elixir may contain the active componentsucrose as a sweetening agent methyl and propylparabens aspreservatives, a dye and flavoring, such as cherry or orange flavor. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compounds may be incorporated intosustained-release preparation and formulations.

Typically, these formulations may contain at least about 0.1% of theactive compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 60% or 70% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound(s) ineach therapeutically useful composition may be prepared is such a waythat a suitable dosage will be obtained in any given unit dose of thecompound. Factors such as solubility, bioavailability, biologicalhalf-life, route of administration, product shelf life, as well as otherpharmacological considerations will be contemplated by one skilled inthe art of preparing such pharmaceutical formulations, and as such, avariety of dosages and treatment regimens may be desirable.

Therapeutics administered by the oral route can often be alternativelyadministered by the lower enteral route, i.e., through the anal portalinto the rectum or lower intestine. Rectal suppositories, retentionenemas or rectal catheters can be used for this purpose and may bepreferred when patient compliance might otherwise be difficult toachieve (e.g., in pediatric and geriatric applications, or when thepatient is vomiting or unconscious). Rectal administration may result inmore prompt and higher blood levels than the oral route, but theconverse may be true as well (Harvey, 1990). Because about 50% of thetherapeutic that is absorbed from the rectum will bypass the liver,administration by this route significantly reduces the potential forfirst-pass metabolism (Benet et al., 1996).

The term “parenteral delivery” refers to the administration of atherapeutic of the invention to an animal, patient or subject in amanner other than through the digestive canal. Means of preparing andadministering parenteral pharmaceutical compositions are known in theart (see, e.g., Avis, 1990).

Intraluminal administration, for the direct delivery of a therapeutic toan isolated portion of a tubular organ or tissue (e.g., such as anartery, vein, ureter or urethra), may be desired for the treatment ofpatients with diseases or conditions afflicting the lumen of such organsor tissues. To effect this mode of administration, a catheter or cannulais surgically introduced by appropriate means. After isolation of aportion of the tubular organ or tissue for which treatment is sought, acomposition comprising a therapeutic of the invention is infused throughthe cannula or catheter into the isolated segment. After incubation forfrom about 1 to about 120 minutes, during which the therapeutic is takenup or in contact with the cells of the interior lumen of the vessel, theinfusion cannula or catheter is removed and flow within the tubularorgan or tissue is restored by removal of the ligatures which effectedthe isolation of a segment thereof (Morishita et al., 1993). Therapeuticcompositions of the invention may also be combined with a biocompatiblematrix, such as a hydrogel material, and applied directly to vasculartissue in vivo.

Intraventricular administration, for the direct delivery of atherapeutic to the brain of a patient, may be desired for the treatmentof patients with diseases or conditions afflicting the brain. One methodto affect this mode of administration, a silicon catheter is surgicallyintroduced into a ventricle of the brain of a human patient, and isconnected to a subcutaneous infusion pump (Medtronic Inc., Minneapolis,Minn.) that has been surgically implanted in the abdominal region (Zimmet al., 1984; Shaw, 1993). The pump is used to inject the therapeuticand allows precise dosage adjustments and variation in dosage scheduleswith the aid of an external programming device. The reservoir capacityof the pump is 18-20 mL and infusion rates may range from 0.1 mL/h to 1mL/h. Depending on the frequency of administration, ranging from dailyto monthly, and the dose of drug to be administered, ranging from0.01-100 microgram per kg of body weight, the pump reservoir may berefilled at 3-10 week intervals. Refilling of the pump may beaccomplished by percutaneous puncture of the self-sealing septum of thepump.

Intrathecal drug administration, for the introduction of a therapeuticinto the spinal column of a patient may be desired for the treatment ofpatients with diseases of the central nervous system. To effect thisroute of administration, a silicon catheter may be surgically implantedinto the L3-4 lumbar spinal interspace of a human patient, and isconnected to a subcutaneous infusion pump which has been surgicallyimplanted in the upper abdominal region (Luer and Hatton, 1993; Ettingeret al., 1978; Yaida et al., 1995). The pump is used to inject thetherapeutic and allows precise dosage adjustments and variations in doseschedules with the aid of an external programming device. Theadministered dose may be similar to that for intraventricularadministration.

To effect delivery to areas other than the brain or spinal column viathis method, the silicon catheter is configured to connect thesubcutaneous infusion pump to, e.g., the hepatic artery, for delivery tothe liver (Kemeny et al., 1993).

Vaginal delivery provides local treatment and avoids first passmetabolism, degradation by digestive enzymes, and potential systemicside-effects. Vaginal suppositories (Remington's PharmaceuticalSciences, 18th Ed., 1990) or topical ointments can be used to effectthis mode of delivery.

In certain embodiments, the inventors contemplate the use of liposomes,nanocapsules, microparticles, microspheres, lipid particles, vesicles,and the like, for the introduction of the compositions of the presentinvention into suitable host cells. In particular, the compositions ofthe present invention may be formulated for delivery either encapsulatedin a lipid particle, a liposome, a vesicle, a nanosphere, or ananoparticle or the like.

Such formulations may be preferred for the introduction ofpharmaceutically-acceptable formulations of the nucleic acids orconstructs disclosed herein. The formation and use of liposomes isgenerally known to those of skill in the art (see for example, Couvreuret al., 1977; Lasic, 1998; which describes the use of liposomes andnanocapsules in the targeted antibiotic therapy for intracellularbacterial infections and diseases). Recently, liposomes were developedwith improved serum stability and circulation half-times (Gabizon andPapahadjopoulos, 1988; Allen and Choun, 1987; U.S. Pat. No. 5,741,516,specifically incorporated herein by reference in its entirety). Further,various methods of liposome and liposome like preparations as potentialdrug carriers have been reviewed (Takakura, 1998; Chandran et al., 1997;Margalit, 1995; U.S. Pat. Nos. 5,567,434; 5,552,157; 5,565,213;5,738,868 and 5,795,587, each specifically incorporated herein byreference in its entirety).

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 .mu.m. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 ANG, containing an aqueous solution in the core.

The fate and disposition of intravenously injected liposomes depend ontheir physical properties, such as size, fluidity, and surface charge.They may persist in tissues for h or days, depending on theircomposition, and half lives in the blood range from min to several h.Larger liposomes, such as MLVs and LUVs, are taken up rapidly byphagocytic cells of the reticuloendothelial system, but physiology ofthe circulatory system restrains the exit of such large species at mostsites. They can exit only in places where large openings or pores existin the capillary endothelium, such as the sinusoids of the liver orspleen. Thus, these organs are the predominant site of uptake. On theother hand, SUVs show a broader tissue distribution but still aresequestered highly in the liver and spleen. In general, this in vivobehavior limits the potential targeting of liposomes to only thoseorgans and tissues accessible to their large size. These include theblood, liver, spleen, bone marrow, and lymphoid organs.

Alternatively, the invention provides for pharmaceutically-acceptablenanocapsule formulations of the compositions of the present invention.Nanocapsules can generally entrap compounds in a stable and reproducibleway (Baszkin et al., 1987; Quintanar-Guerrero et al., 1998; Douglas etal., 1987). To avoid side effects due to intracellular polymericoverloading, such ultrafine particles (sized around 0.1 .mu.m) should bedesigned using polymers able to be degraded in vivo. Biodegradablepolyalkyl-cyanoacrylate nanoparticles that meet these requirements arecontemplated for use in the present invention. Such particles may beeasily made, as described (Couvreur et al., 1980; 1988; zur Muhlen etal., 1998; Zambaux et al. 1998; Pinto-Alphandry et al., 1995 and U.S.Pat. No. 5,145,684, specifically incorporated herein by reference in itsentirety).

E. Treatment of Pathophysiological States 1. Definitions

A “pathophysiological state” is defined herein to refer to a disease orhealth-related condition. “Treatment” and “treating” as used hereinrefer to administration or application of a therapeutic agent to asubject or performance of a procedure or modality on a subject for thepurpose of obtaining a therapeutic benefit of a disease orhealth-related condition. For example, a therapeutic polypeptide of thepresent invention can be administered for the purpose of reducingtemperature in a patient with high body temperature or reducing symptomsof congestive heart failure in a patient with congestive heart failure.

The term “therapeutic benefit” or “therapeutically effective” as usedthroughout this application refers to anything that promotes or enhancesthe well-being of the subject with respect to the medical treatment ofthis condition. This includes, but is not limited to, a reduction in thefrequency or severity of the signs or symptoms of a disease.

“Prevention” and “preventing” are used according to their ordinary andplain meaning to mean “acting before” or such an act. In the context ofa particular disease or health-related condition, those terms refer toadministration or application of an agent, drug, or remedy to a subjector performance of a procedure or modality on a subject for the purposeof blocking the onset of a disease or health-related condition.

2. Pathophysiological States to be Treated or Prevented

The polypeptides and polynucleotides of the present invention can beapplied in the treatment or prevention of any disease or health-relatedcondition. The disease or health-related condition can be any disease orhealth-related condition for which administration of the polypeptides orpolynucleotides of the present invention are known or suspected to be ofvalue. Examples include, but are not limited to high body temperature,appetite dysfunction, congestive heart failure, stress, anxiety, andundesirably low levels of ACTH secretion.

3. Secondary Treatment

Certain embodiments of the present invention provide for theadministration or application of one or more secondary forms oftherapies for the treatment or prevention of a pathophysiological state.

The secondary form of therapy may be administration of one or moresecondary pharmacological agents that can be applied in the treatment orprevention of a pathophysiological state.

If the secondary therapy is a pharmacological agent, it may beadministered prior to, concurrently, or following administration of thepolypeptide or polynucleotide of the present invention.

The interval between the polypeptide or polynucleotide of the presentinvention and the secondary therapy may be any interval as determined bythose of ordinary skill in the art. For example, the interval may beminutes to weeks. In embodiments where the agents are separatelyadministered, one would generally ensure that a significant period oftime did not expire between the time of each delivery, such that eachtherapeutic agent would still be able to exert an advantageouslycombined effect on the subject. For example, the interval betweentherapeutic agents may be about 12 h to about 24 h of each other and,more preferably, within about 6 hours to about 12 h of each other. Insome situations, it may be desirable to extend the time period fortreatment significantly, however, where several d (2, 3, 4, 5, 6 or 7)to several wk (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respectiveadministrations. In some embodiments, the timing of administration of asecondary therapeutic agent is determined based on the response of thesubject to the the polypeptide or polynucleotide of the presentinvention.

F. Kits and Diagnostics

In various aspects of the invention, a kit is envisioned containing oneor more polypeptides or polynucleotides of the present invention. Insome embodiments, the present invention contemplates a kit for preparingand/or administering a therapy of the invention. The kit may compriseone or more sealed vials containing any of the pharmaceuticalcompositions of the present invention. In some embodiments, the kit mayalso comprise a suitable container means, which is a container that willnot react with components of the kit, such as an eppendorf tube, anassay plate, a syringe, a bottle, or a tube. The container may be madefrom sterilizable materials such as plastic or glass.

The kit may further include an instruction sheet that outlines one ormore methods of the invention, and will follow substantially the sameprocedures as described herein or are known to those of ordinary skill.The instruction information may be in a computer readable mediacontaining machine-readable instructions that, when executed using acomputer, cause the display of a real or virtual procedure of deliveringa pharmaceutically effective amount of a therapeutic agent.

G. Transgenic Animals

Transgenic animals and cell lines derived from such animals may find usein certain testing experiments. In one embodiment of the invention,transgenic animals are produced which contain a functional transgeneencoding a polypeptide of the present invention. Such transgenic animalsmay be useful in methods for identifying additional therapeuticapplications of the claimed polypeptides. Transgenic animals of thepresent invention also can be used as models for studying indications.

In one embodiment of the invention, a transgene is introduced into anon-human host to produce a transgenic animal expressing a polypeptideof the present invention. The transgenic animal is produced by theintegration of the transgene into the genome in a manner that permitsthe expression of the transgene. Methods for producing transgenicanimals are generally described by Wagner and Hoppe (U.S. Pat. No.4,873,191; which is incorporated herein by reference), Brinster et al.(1985); which is incorporated herein by reference in its entirety) andin “Manipulating the Mouse Embryo; A Laboratory Manual” (1994); which isincorporated herein by reference in its entirety).

Transgenic animals may be produced by any method known to those ofordinary skill in the art. For example, they can be produced from thefertilized eggs from a number of animals including, but not limited toreptiles, amphibians, birds, mammals, and fish. Within a particularembodiment, transgenic mice are generated which overexpress apolypeptide of the present invention.

H. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Identification of Biologically Active Urocortin 2 Polypeptides

Urocortin 2 precursors were overexpressed in various cell lines, andtheir function was evaluated. Pancreatic and skin cell lines wereinfected with mouse urocortin 2 (mUcn 2) lentivirus. The amino acidsequence of mouse urocortin 2 is provided as SEQ ID NO:10. The cellswere found to secrete a precursor and a smaller processed zone whichcontained a urocortin peptide: R⁰mUcn 2 (SEQ ID NO:11) which wasC-terminally amidated. The peptide was synthesized and bioactivity wasdetermined by the stimulation of cAMP accumulation in A7r5 cells, whichexpress CRFR2. The R⁰mUcn 2 peptide was found to be equipotent to mUcn2(1-38), with EC_(50S) of 0.05 (0.028-0.1)² nM and 0.07 (0.03-0.15)¹¹nM, respectively. It is possible that R⁰mUcn 2 could be furtherprocessed by aminopeptidases to mUcn 2(1-38). Number of assays aredepicted as a superscript.

The corresponding human analog, R⁰hUcn 2, was synthesized. This peptidewas found to be equipotent to hUcn 2(1-38) in the ability to stimulatecAMP accumulation in A7r5 cells, with EC_(50S) of 0.027 (0.023-0.031)²nM and 0.11 (0.058-0.21)¹¹ nM, respectively.

A peptide extended with a hydrophilic arginine residue such as R⁰hUcn 2is likely more soluble, and therefore, a better pharmaceutical agent,than hUcn 2(1-38). As evidence of increased solubility, QC reverse phaseHPLC using a trifluoroacetic acid/acetonitrile solvent system showed theretention time of hUcn 2(1-38) was 16.9 minutes whereas the retentiontime of R⁰hUcn 2 was 13.9 minutes, demonstrating significantly increasedhydrophilicity for the arginine extended analog. Table 2 providessequence information of selected Ucn 2 sequences.

TABLE 2 Sequence Information Sequence Peptide Identifier SequencehUcn 2-OH SEQ ID IVLSLDVPIGLLQILLEQARARAAREQATTNARILARV NO: 1 hUcn 2-G-SEQ ID IVLSLDVPIGLLQILLEQARARAAREQATTNARILARV OH NO: 2 G hUcn 2-GH-SEQ ID IVLSLDVPIGLLQILLEQARARAAREQATTNARILARV OH NO: 3 GH hUcn 2-GHC-SEQ ID IVLSLDVPIGLLQILLEQARARAAREQATTNARILARV OH NO: 4 GHC hUcn 2 SEQ IDIVLSLDVPIGLLQILLEQARARAAREQATTNARILARV- NO: 5NH. sub.2 (amidated at the C-terminus) R⁰ - hUcn 2 SEQ IDRIVLSLDVPIGLLQILLEQARARAAREQATTNARILAR NO: 6V-NH.sub.2 (amidated at the C-terminus) SRP SEQ IDHPGSRIVLSLDVPIGLLQILLEQARARAAREQATTNARI (HPGSR- NO: 7LARV-NH.sub.2 (amidated at the C-terminus) hUcn 2) [Cys^(1,51) SEQ IDCSPTRHPGSRIVLSLDVPIGLLQILLEQARARAAREQAT (Acm)]-hUcn NO: 8 TNARILARVGHC2(1-51)-OH) hUcn 2 SEQ ID IPTFQLRPQNSPQTTPRPAASESPSAAPTWPWAAQSHCprohormone NO: 9 SPTRHPGSRIVLSLDVPIGLLQILLEQARARAAREQATTNARILARV-NH2(amidated at the C-terminus) mUcn2 - SEQ IDVILSLDVPIGLLRILLEQARYKAARNQAATNAQILAHV OH NO: 21 mUcn 2 SEQ IDVILSLDVPIGLLRILLEQARYKAARNQAATNAQILAHV NO: 10(amidated at the C-terminus) R⁰ - mUcn 2 SEQ IDRVILSLDVPIGLLRILLEQARYKAARNQAATNAQILAH NO: 11V (amidated at the C-terminus) mUcn 2 SEQ IDRVILSLDVPIGLLRILLEQARYKAARNQAATNAQILAH prohormone- NO: 22 V OH mUcn 2SEQ ID TPIPTFQLLPQNSLETTPSSVTSESSSGTTTGPSASWSNS prohormone NO: 12KASPYLDTRVILSLDVPIGLLRILLEQARYKAARNQAATNAQILAHV (amidated at the C-terminus) R⁰ - hUcn 2 - SEQ IDRIVLSLDVPIGLLQILLEQARARAAREQATTNARILAR G-OH NO: 13 VG R⁰ - hUcn 2-SEQ ID RIVLSLDVPIGLLQILLEQARARAAREQATTNARILAR GH-OH NO: 14 VGHR⁰ - hUcn 2- SEQ ID RIVLSLDVPIGLLQILLEQARARAAREQATTNARILAR GHC-OH NO: 15VGHC hUcn 2 SEQ ID IPTFQLRPQNSPQTTPRPAASESPSAAPTWPWAAQSHC prohormone -NO: 16 SPTRHPGSRIVLSLDVPIGLLQILLEQARARAAREQATT G-OH NARILARVG hUcn 2SEQ ID IPTFQLRPQNSPQTTPRPAASESPSAAPTWPWAAQSHC prohormone - NO: 17SPTRHPGSRIVLSLDVPIGLLQILLEQARARAAREQATT GH-OH NARILARVGH hUcn 2 SEQ IDIPTFQLRPQNSPQTTPRPAASESPSAAPTWPWAAQSHC prohormone - NO: 18SPTRHPGSRIVLSLDVPIGLLQILLEQARARAAREQATT GHC-OH NARILARVGHC hUcn 2 SEQ IDIPTFQLRPQNSPQTTPRPAASESPSAAPTWPWAAQSHC prohormone- NO: 19SPTRHPGSRIVLSLDVPIGLLQILLEQARARAAREQATT OH NARILARV

Table 3 shows a comparison of amino acid sequences and bioactivity ofselected synthetic human or human Ucn 2 peptide analogues. The EC_(50S)were calculated based on protein determination by immunoassay andchromatographic peak height.

TABLE 3 Bioactivity of Selected Synthetic Human or Mouse Ucn 2Peptides/Analogues cAMP A7r5 SEQUENCE (EC₅₀, nM) [Number of PeptideIDENTIFIER Assays] hUcn 2-OH SEQ ID NO: 1 90.4 (47-172) [3] hUcn 2-G-OHSEQ ID NO: 2 6.3 (3.4-11.5) [2] hUcn 2-GH-OH SEQ ID NO: 3 2.9 (0.93-9.1)[2] hUcn 2-GHC-OH SEQ ID NO: 4 13.9 (11.3-17) [2] hUcn 2 SEQ ID NO: 50.11 (0.058-0.21) [11] R⁰-hUcn 2 SEQ ID NO: 6 0.027 (0.023-0.031) [2]SRP (HPGSR-hUcn 2) SEQ ID NO: 7 0.09 (0.033-0.22) [2] [Cys^(1,51)(Acm)]-hUcn 2(1- SEQ ID NO: 8 9.5 (3.3-26.8) [3] 51)-OH) hUcn 2prohormone SEQ ID NO: 9 2.3 (1.8-3) [2] mUcn 2 SEQ ID NO: 10 0.07(0.03-0.15) [11] R⁰-mUcn 2 SEQ ID NO: 11 0.05 (0.03-0.1) [2] mUcn 2prohormone SEQ ID NO: 12 0.53 (0.4-0.8) [2]

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe methods of this invention have been described in terms of preferredembodiments, it will be apparent to those of skill in the art thatvariations may be applied to the methods described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1-36. (canceled)
 37. A Urocortin 2 polypeptide or peptide derived fromUrocortin 2, or a polypeptide variant thereof.
 38. A Urocortin 2polypeptide or peptide derived from a prohormone of Urocortin 2 or apolypeptide variant thereof.
 39. The polypeptide or peptide of claim 37or 38, wherein the C-terminus is amidated.
 40. The polypeptide orpeptide of claim 37, wherein the polypeptide or peptide is modified. 41.The polypeptide or peptide of claim 40, wherein the modification is theaddition of a fatty acid or the addition of at least one amino acidresidue.
 42. The polypeptide or peptide of claim 37, wherein the variantis a substitutional or an insertional mutant, wherein an insertionalmutant contains one or more additional amino acid residues.
 43. Thepolypeptide or peptide of claim 38, wherein the variant is asubstitutional or an insertional mutant, wherein an insertional mutantcontains one or more additional amino acid residues.
 44. The polypeptideor peptide of claim 39, wherein the variant is a substitutional or aninsertional mutant, wherein an insertional mutant contains one or moreadditional amino acid residues.
 45. The polypeptide or peptide of claim44, wherein the at least one amino acid residue is selected from R and Kor a combination thereof.
 46. The polypeptide or peptide of claim 37,wherein the N-terminus is modified by acylation or methylation.
 47. Thepolypeptide or peptide of claim 37 or salts thereof.
 48. The polypeptideor peptide of claim 37, wherein the Urocortin 2 is SEQ ID NO: 1 or SEQID NO:
 10. 49. The polypeptide or peptide of claim 37 or 38, wherein thepolypeptide or peptide is selected from the group consisting of SEQ IDNOS: 1-19 and 21-22.
 50. The polypeptide or peptide of claim 37, whereinthe variant is a chimeric protein, a fusion protein or a chemicalderivative.
 51. A composition comprising the polypeptide or peptide ofclaim 37 in a pharmaceutically acceptable carrier.
 52. The compositionof claim 51, wherein the pharmaceutical form is formulated forparenteral administration, for oral administration, or for injection viathe intravenous, intramuscular, subcutaneous, or intraperitoneal routes.53. A method of treating congestive heart failure, low levels of ACTHsecretion or abnormalities of glucose metabolism comprisingadministering to a subject a composition of claim
 49. 54. The method ofclaim 53, wherein the abnormalities of glucose metabolism are selectedfrom the group consisting of diabetes, impaired glucose tolerance, andhyperglycemia.